Our recent efforts have examined novel activating Ly-49 NK receptors. Murine Ly-49D and Ly-49H augments NK cell function upon recognition of target cells. In addition, we demonstrate that Ly-49D and Ly49H receptor ligation can lead to the rapid and potent secretion of IFN-g. Cytokine secretion can be induced from NK cells after receptor ligation with Ab or after interaction with target cells expressing their H-2Dd ligand. Consistent with the dominant inhibitory function of Ly-49G, NK cells coexpressing Ly-49D and Ly-49G show a profound reduction in IFN-g secretion after interaction with targets expressing their common ligand. It is well known that NK cells expressing both receptors are presented with a ligand, inhibition dominates the functional outcome. We have demonstrated that costimulation of the activating Ly-49D murine NK cell receptor with IL-12 or IL-18 is capable of over-riding the inhibitory Ly-49G2 receptor blockade for cytokine production both in vitro and in vivo. This synergy is mediated by and dependent upon Ly-49D-expressing NK cells and results in significant systemic expression of IFN-gamma. This would place NK cells and their activating Ly-49 receptors as important initiators of microbial, antiviral, and antitumor immunity and provide a mechanism for the release of activating Ly-49 receptors from inhibitory receptor blockade. Our recent studies have extended these co-stimulatory events to other ITAM associated NK receptors (NKRs). Examination of NKRp1 and NKG2D demonstrated parallel and similar result to Ly-49D, in that co-stimulation with IL-12 or IL-18 resulted in both co-stimulation of IFN-g secretion as well as release from dominant inhibitory NKRs. Finally, this IL-12 and IL-18 co-stimulation was examined on T cells and was also found to be a relevant co-stimuli for the T cell receptor for production of IFN-g secretion. Thus inflammatory signals in vivo can have potent activating co-stimulatory effects on immune functions that would normally be down regulated in vivo. NKT and NK cells are important immune regulatory cells. The only efficient means to selectively stimulate NKT cells in vivo is alpha-galactosylceramide (alphaGalCer). However, alphaGalCer effectively stimulates and then diminishes the number of detectable NKT cells. It also exhibits a potent, indirect ability to activate NK cells. We have reported on a novel ceramide compound, beta-galactosylceramide (betaGalCer) (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies using CD1d tetramers loaded with betaGalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared with alphaGalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, betaGalCer (C12), in contrast to alphaGalCer, failed to increase NK cell size, number, and cytolytic activity. Also in contrast to alphaGalCer, betaGalCer (C12) is a poor inducer of IFN-gamma, TNF-alpha, GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, alphaGalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while betaGalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show betaGalCer (C12) can effectively discriminate between NKT- and NK-mediated responses in vivo. These results indicate the use of different TCR-binding ceramides can provide a unique approach for understanding the intricate immunoregulatory contributions of these two cell types.